ABSTRACT Using human induced pluripotent stem cells (iPSCs) as a source of platelets (Plt)s for transfusion medicine is being pursued by a number of groups with several proposing that such a product will soon be available. To achieve this goal, it would be important to generate a Plt product that overcomes challenges associated with donors Plts, including the issue that iPSC-derived hematopoietic progenitors tend to be primitive in nature and the subsequent megakaryocytes, termed Prim-Megs, and the platelets they produce are primitive as well with many quantitative and qualitative limitations due to this immature developmental stage. In our parent R01 application, we follow a number of strategies to define the proper hematopoietic progenitor to generate the highest quality of megakaryocyte with the potential to generate the highest yield and function of derivative Plts. We have focused in the parent grant on obtaining hematopoietic lineages of later developmental stages collectively called definitive hematopoiesis that can generate definitive megakaryocytes, termed Def-Megs. These cells are distinct and have differing abilities to generate Plts with appropriate biologic activity than Prim- Megs. While our parent R01 aims to compare these two populations generated from pluripotent stem cells and optimize the generation of the highest quality megakaryocyte possible, we did not address a final maturation event that occurs after birth: While both neonatal and adult hematopoietic progenitors are definitive and are derived from the same developmental program, the megakaryocytes derived from these populations are distinct. Neonate-Megs are low ploidy and generate Plts with poor activity in clotting assays compared to adult- Megs. Recent work suggests that the RNA binding protein IGF2BP3 may regulate this maturation process. Its expression is lost in adult-Megs and suppression of IGF2BP3 in cord blood neonate-Megs results in maturation more similar to adult-Megs. We show preliminary data that iPSC-derived megakaryocytes (iMegs) also express high levels of IGF2BP3 and its suppression results in higher ploidy megakaryocytes. We now wish to apply these findings to enhance maturation of iMegs in the following 2 Specific Aims (SAs): SA#1: To determine the effect of IGF2BP3 inhibition on maturation of iMegs in vitro. We will utilize various methods to inhibit IGF2BP3 in iMegs and examine the impact on megakaryocyte maturation in vitro. SA#2: To determine the effect of IGF2BP3 inhibition on release of platelets from iMegs in vivo. Using the same methodologies to inhibit IGF2BP3 as in SA#1, we will examine the ability of the megakaryocytes to function in a mouse xeno- infusion model, examining Plt yield, half-life and ability to contribute to clot formation. We believe that these studies will not only be important in bringing such Plts closer to clinical application, but serve as a role model of how one can improve regenerative medicine in general by obtaining adult rather than less functional embryonic tissues.